WO2021217773A1 - Transaminase mutant and use thereof - Google Patents
Transaminase mutant and use thereof Download PDFInfo
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- WO2021217773A1 WO2021217773A1 PCT/CN2020/093528 CN2020093528W WO2021217773A1 WO 2021217773 A1 WO2021217773 A1 WO 2021217773A1 CN 2020093528 W CN2020093528 W CN 2020093528W WO 2021217773 A1 WO2021217773 A1 WO 2021217773A1
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- 102000003929 Transaminases Human genes 0.000 title claims abstract description 42
- 108090000340 Transaminases Proteins 0.000 title claims abstract description 42
- 230000035772 mutation Effects 0.000 claims abstract description 36
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 19
- 150000001412 amines Chemical class 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims description 27
- -1 ketone compound Chemical class 0.000 claims description 21
- 210000004027 cell Anatomy 0.000 claims description 19
- 108020004414 DNA Proteins 0.000 claims description 18
- 239000013612 plasmid Substances 0.000 claims description 17
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 9
- 238000005891 transamination reaction Methods 0.000 claims description 9
- 102000053602 DNA Human genes 0.000 claims description 8
- 210000001236 prokaryotic cell Anatomy 0.000 claims description 7
- 229910052736 halogen Inorganic materials 0.000 claims description 6
- KWOLFJPFCHCOCG-UHFFFAOYSA-N Acetophenone Natural products CC(=O)C1=CC=CC=C1 KWOLFJPFCHCOCG-UHFFFAOYSA-N 0.000 claims description 5
- 241000588724 Escherichia coli Species 0.000 claims description 5
- 229910052731 fluorine Inorganic materials 0.000 claims description 5
- 125000001153 fluoro group Chemical group F* 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 5
- 150000008062 acetophenones Chemical class 0.000 claims description 4
- 150000002367 halogens Chemical class 0.000 claims description 4
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 abstract description 34
- 239000000758 substrate Substances 0.000 abstract description 12
- 150000002576 ketones Chemical class 0.000 abstract description 9
- 230000003197 catalytic effect Effects 0.000 abstract description 8
- 230000001976 improved effect Effects 0.000 abstract description 5
- 238000009776 industrial production Methods 0.000 abstract description 5
- 239000002994 raw material Substances 0.000 description 31
- 230000000694 effects Effects 0.000 description 30
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- 102000004190 Enzymes Human genes 0.000 description 14
- 108090000790 Enzymes Proteins 0.000 description 14
- 238000003752 polymerase chain reaction Methods 0.000 description 14
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- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- 102000039446 nucleic acids Human genes 0.000 description 7
- 108020004707 nucleic acids Proteins 0.000 description 7
- 150000007523 nucleic acids Chemical class 0.000 description 7
- ISYORFGKSZLPNW-UHFFFAOYSA-N propan-2-ylazanium;chloride Chemical compound [Cl-].CC(C)[NH3+] ISYORFGKSZLPNW-UHFFFAOYSA-N 0.000 description 7
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 description 7
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 6
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 125000000217 alkyl group Chemical group 0.000 description 6
- 125000003710 aryl alkyl group Chemical group 0.000 description 6
- 125000003118 aryl group Chemical group 0.000 description 6
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- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 6
- 238000006467 substitution reaction Methods 0.000 description 5
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- 125000005843 halogen group Chemical group 0.000 description 4
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 4
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- LCEINQHGZAFADC-UHFFFAOYSA-N 1-(4-methylpyridin-3-yl)ethanone Chemical compound CC(=O)C1=CN=CC=C1C LCEINQHGZAFADC-UHFFFAOYSA-N 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
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- 238000003786 synthesis reaction Methods 0.000 description 3
- 229940113082 thymine Drugs 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- SUGXZLKUDLDTKX-UHFFFAOYSA-N 1-(2-nitrophenyl)ethanone Chemical compound CC(=O)C1=CC=CC=C1[N+]([O-])=O SUGXZLKUDLDTKX-UHFFFAOYSA-N 0.000 description 2
- HCEKGPAHZCYRBZ-UHFFFAOYSA-N 1-(3-fluorophenyl)ethanone Chemical compound CC(=O)C1=CC=CC(F)=C1 HCEKGPAHZCYRBZ-UHFFFAOYSA-N 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
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- 102220591179 Receptor-type tyrosine-protein phosphatase kappa_H82S_mutation Human genes 0.000 description 2
- 229960000643 adenine Drugs 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 description 2
- 125000004093 cyano group Chemical group *C#N 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 125000001301 ethoxy group Chemical group [H]C([H])([H])C([H])([H])O* 0.000 description 2
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- 229910052717 sulfur Inorganic materials 0.000 description 2
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- 241000589158 Agrobacterium Species 0.000 description 1
- 108020005544 Antisense RNA Proteins 0.000 description 1
- RQEUFEKYXDPUSK-SSDOTTSWSA-N C[C@H](c1ccccc1)N Chemical compound C[C@H](c1ccccc1)N RQEUFEKYXDPUSK-SSDOTTSWSA-N 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
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- 241000672609 Escherichia coli BL21 Species 0.000 description 1
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- 241000192125 Firmicutes Species 0.000 description 1
- 229930194542 Keto Natural products 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 108020004417 Untranslated RNA Proteins 0.000 description 1
- 102000039634 Untranslated RNA Human genes 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
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- 239000002220 antihypertensive agent Substances 0.000 description 1
- 229940127088 antihypertensive drug Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 229940125692 cardiovascular agent Drugs 0.000 description 1
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- JBAXWZGBNPCMNR-UHFFFAOYSA-N hexan-2-one Chemical compound CCCCC(C)=O.CCCCC(C)=O JBAXWZGBNPCMNR-UHFFFAOYSA-N 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 150000002466 imines Chemical class 0.000 description 1
- 125000001841 imino group Chemical group [H]N=* 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000006317 isomerization reaction Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 125000000468 ketone group Chemical group 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 230000000926 neurological effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
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- 150000003624 transition metals Chemical class 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/001—Amines; Imines
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/008—Preparation of nitrogen-containing organic compounds containing a N-O bond, e.g. nitro (-NO2), nitroso (-NO)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
- C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y206/00—Transferases transferring nitrogenous groups (2.6)
- C12Y206/01—Transaminases (2.6.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Definitions
- the invention relates to the field of enzyme production, in particular to a transaminase mutant and its application.
- Chiral amines are the structural units of many important biologically active molecules and are key intermediates for the synthesis of many chiral drugs. Many important neurological drugs, cardiovascular drugs, antihypertensive drugs, anti-infective drugs, etc. are synthesized using chiral amines as intermediates. The preparation of chiral amines is mainly achieved through chemical reduction methods. There are disadvantages such as harsh reaction conditions, the use of toxic transition metal catalysts, and low product stereoselectivity. Currently, the use of biocatalysis to prepare chiral compounds in the pharmaceutical industry is widely used and has broad prospects.
- Omega-transaminase can catalyze ketone substrates and efficiently prepare chiral amines through stereoselective transamination. Because of its high selectivity, high conversion rate and mild reaction conditions, it has received extensive attention from researchers. However, in industrial applications, most of the wild transaminases have shortcomings such as low catalytic efficiency, poor stereoselectivity, and weak stability, so that there are not many transaminase that can really be used.
- the invention patent application with application publication number CN107828751A discloses an ⁇ -transaminase mutant L107I derived from Actinobacteria sp., which can highly selectively catalyze acetophenone compounds to obtain chiral ammonia products.
- the reaction is as follows:
- the initial activity is less than 10%, the activity is low, the amount of enzyme added during the reaction is large, and the post-processing is difficult.
- the main purpose of the present invention is to provide a transaminase mutant and its application to solve the problem that the existing transaminase is not suitable for industrial production.
- a transaminase mutant is provided. Compared with the amino acid sequence shown in SEQ ID NO:1, the amino acid sequence of the transaminase mutant includes at least one mutation site as follows: L166 , K149, K146, A168, H73, F133, H82, E24, V194, T294, A295, G235 and F236.
- amino acid sequence of the aminotransferase mutant includes at least one mutation site as follows: L166I/V, K149Q/V/C/I/W/M/Y/H/L/F/R, K146R/M/A/P , A168M/V/I/S/P/F, H73T/N/C/Q/R/W/M/K/S, F133S/Q/M/R/A/D, H82S ⁇ Q ⁇ E ⁇ T ⁇ Y, E24V/S/A/F, V194I/S/A/H/N, T294Q/W/V/A/E/R/Y/F, A295S/Y/I/M/G, G235Y/H And F236I/T/P/M/V/D/S, Y9N, S132K, R145K/P/S/M/Y, L151R/M/A, T178M, "/" means "or”; or aminotransferase mutants include
- amino acid sequence of the aminotransferase mutant has any combination of mutation sites in the following table:
- a DNA molecule which encodes any of the above-mentioned aminotransferase mutants.
- a recombinant plasmid is provided, which is connected with any of the above-mentioned DNA molecules.
- a host cell which contains any of the above recombinant plasmids.
- the host cell includes a prokaryotic cell or a eukaryotic cell; preferably, the prokaryotic cell is Escherichia coli.
- a method for producing chiral amines includes: using any of the above-mentioned aminotransferase mutants to catalyze ketone compounds Carry out transamination reaction to produce product
- R 1 and R 2 each independently represent an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group
- R 1 and R 2 can be singly or combined with each other to form a substituted or unsubstituted ring.
- R 1 and R 2 are an optionally substituted or unsubstituted alkyl group having 1 to 20 carbon atoms, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group.
- R 1 and R 2 are an optionally substituted or unsubstituted alkyl group having 1-10 carbon atoms, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted Aryl; preferably, substitution refers to a halogen atom, a nitrogen atom, a sulfur atom, a hydroxyl group, a nitro group, a cyano group, a methoxy group, an ethoxy group, a carboxyl group, a carboxymethyl group, a carboxyethyl group or a methylenedioxy group replace.
- the ketone compound is Or the ketone compound is an acetophenone compound substituted or unsubstituted at any position on the benzene ring, catalyzed by the aminotransferase mutant
- Carry out transamination reaction to produce product R represents that any position on the benzene ring is substituted by halogen, nitro, methyl or methoxy; preferably, the halogen is a fluorine atom; more preferably, the fluorine atom is substituted at the meta position of the benzene ring; preferably, the nitro group is substituted on the benzene ring Ortho substitution; preferably, acetophenone compounds are
- the mutant of the present invention has improved catalytic activity for transamination of ketone substrates, and is suitable for the industrial production of chiral amines.
- Naturally occurring or wild type is the opposite of “mutant” and refers to the form found in nature.
- a naturally-occurring or wild-type polypeptide or polynucleotide sequence is a sequence that exists in an organism, it can be isolated from a source in nature, and has not been intentionally modified or changed by man.
- the "recombination" of cells, nucleic acids, or polypeptides when referring to, for example, the "recombination" of cells, nucleic acids, or polypeptides, it means that they have been modified in a way that does not exist in nature, or are in the same form as they exist in nature, but are made of synthetic materials and/or through the use of recombinant materials. It is prepared or derived by technological processing, or corresponds to a cell, nucleic acid or polypeptide in a natural or inherent form. Among them, non-limiting examples include recombinant cells expressing genes other than the inherent (non-recombinant) form in cells or expressing inherent genes at different levels.
- Percentage of sequence identity refers to the comparison between polynucleotide sequences or amino acid sequences, and is determined by comparing two optimally aligned sequences across a comparison window, where the polynucleotide sequence or amino acid sequence is in the comparison window. Compared with the reference sequence, the part in may include additions or deletions (ie, gaps) for the best alignment of the two sequences. The percentage can be calculated as follows: by determining the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and The result is multiplied by 100 to get the percentage of sequence identity.
- the percentage can be calculated as follows: by determining the number of positions where the same nucleic acid base or amino acid residue or the nucleic acid base or amino acid residue is aligned with the gap in the two sequences to generate the number of matching positions, the match Divide the number of positions by the total number of positions in the comparison window and multiply the result by 100 to get the percentage of sequence identity.
- reference sequence refers to a designated sequence used as a basis for sequence comparison. The reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence.
- Site-directed mutagenesis refers to the introduction of desired changes (usually to characterize changes in a favorable direction) into the target DNA fragment (either genome or plasmid) by polymerase chain reaction (PCR) and other methods, including the addition of bases , Deletion, point mutation, etc. Site-directed mutagenesis can quickly and efficiently improve the traits and characterization of the target protein expressed by DNA, which is a very useful method in gene research.
- the directed evolution of enzymes is a kind of irrational design of proteins, artificially creating special evolutionary conditions, simulating natural evolutionary mechanisms, modifying genes in vitro, applying error-prone PCR, DNA shuffling and other technologies, combined with efficient screening systems to obtain New enzymes with expected properties.
- this application uses the method of enzyme evolution to analyze the ⁇ -transaminase mutant L107I (SEQ ID NO:1) derived from Actinobacteria sp. ) To evolve.
- the sequence of the ⁇ -transaminase mutant L107I (SEQ ID NO:1) derived from Actinobacteria sp. is as follows:
- Saturation mutation is a method to obtain mutants in which the target amino acids are replaced by 19 other amino acids in a short time by modifying the coding gene of the target protein. This method is not only a powerful tool for directed modification of proteins, but also an important method for the study of protein structure-function relationships. Saturation mutations can often obtain more ideal evolutionary bodies than single point mutations. For these problems that the site-directed mutation method cannot solve, it is precisely the unique point that the saturation mutation method is good at.
- mutants with increased activity obtained by saturation mutations beneficial amino acid positions can be combined to obtain mutants with better traits.
- the method for constructing double-point mutations in combinatorial mutations is the same as that for single-point mutations, using the whole plasmid PCR method. Simultaneously mutate two or more sites of multiple-point mutations by using overlap extension PCR amplification to obtain a mutated gene containing multiple-point mutations. After the two ends are digested with restriction enzymes, they are ligated to the expression vector and transformed into The Escherichia coli cells were spread on an LB culture dish containing 100 ⁇ g/mL ampicillin and cultured overnight at 37° C. to obtain combined mutants, which were identified by sequencing.
- the mutant plasmid obtained by saturation mutation and combined mutation is transformed into E. coli cells and expressed in E. coli. Then, the crude enzyme solution is obtained by sonicating the cells. The best conditions for inducing expression of transaminase: 25°C, 0.2mM IPTG overnight induction.
- transaminase mutants After obtaining transaminase mutants with greatly increased activity, they are randomly mutated using error-prone PCR methods to construct a high-quality mutant library, develop appropriate high-throughput screening methods, screen the library, and obtain mutations with further increased activity body.
- Error-prone PCR means PCR under error-prone conditions, that is, a PCR technique that easily causes errors in the copied DNA sequence, also known as mismatch PCR or error-prone PCR. Specifically, it refers to the use of low-fidelity TaqDNA polymerase and changing PCR reaction conditions to reduce the fidelity of DNA replication, increase base mismatches during the synthesis of new DNA strands, and cause more point mutations in the amplified products. A method of inducing DNA sequence variation in vitro.
- Error-prone PCR is currently the simplest and most effective in vitro random mutagenesis technique for genes. Its principle is: the isomerization of bases provides the possibility of mismatches.
- the four bases that make up DNA all have tautomers.
- the three oxygen-containing bases of guanine (G), cytosine (C) and thymine (T) have two tautomers: keto and enol.
- Adenine (A) and thymine are two nitrogenous bases, and there are amine and imine tautomers.
- G, C and T mainly exist as ketone structure, and the ratio of enol structure is extremely low.
- the nitrogen atoms on the two nitrogen-containing bases of A and T mainly exist in the state of amino (NH2), and in the state of imino (NH).
- the rate of state existence is extremely low.
- the difference in the position of the hydrogen atom between different isomers and the deviation of the electron cloud at the same position can change the pairing form of the bases, so that mismatches may appear on the copied daughter strands. For example, when thymine exists in a ketone structure, it pairs with adenine, and when it exists in an enol structure, it pairs with guanine. In this way, there are unstable bases where A can be matched with C and T can be matched with G. Yes, resulting in mismatches.
- TaqDNA polymerase has the highest mismatch rate.
- Taq DNA polymerase is the most active one among the heat-resistant DNA polymerases found. It has 5'-3' exonuclease activity, but not 3'-5' exonuclease activity. Therefore, it has a strong effect on certain mononuclear DNA polymerases during synthesis. There is no correction function for nucleotide mismatches, so the probability of mismatches is higher than that of DNA polymerases with 3'-5' proofreading activity.
- the fidelity of DNA polymerase can be reduced by a variety of methods, including the use of 4 different concentrations of dNTP, the addition of Mn 2+ , and the increase of Mg 2+ concentration.
- MnC1 2 is a mutagenic factor of DNA polymerase. Adding Mn 2+ can reduce the specificity of the polymerase to the template and increase the mismatch rate; the unbalanced concentration of 4 dNTPs can increase the probability of base misincorporation and achieve mismatches.
- Mg 2+ has the effect of activating Taq enzyme, increasing the concentration of Mg 2+ to exceed the normal dosage, which can stabilize non-complementary base pairs; increasing the dosage of Taq DNA polymerase and increasing the elongation time of each cycle can increase mismatches Probability of terminal extension; reducing the initial template concentration will increase the proportion of variant templates in subsequent PCR cycles.
- a transaminase mutant is provided.
- the amino acid sequence of the transaminase mutant includes at least one mutation site as follows: L166 , K149, K146, A168, H73, F133, H82, E24, V194, T294, A295, G235 and F236.
- the mutant of the present invention has improved catalytic activity for transamination of ketone substrates, and is suitable for the industrial production of chiral amines.
- the amino acid sequence of the aminotransferase mutant further includes one of the following mutation sites: L166I/V, K149Q/V/C/I/W/M/Y/H/L/F/R, K146R/M/A/P, A168M/V/I/S/P/F, H73T/N/C/Q/R/W/M/K/S, F133S/Q/M/R/A/D, H82S ⁇ Q ⁇ E ⁇ T ⁇ Y, E24V/S/A/F, V194I/S/A/H/N, T294Q/W/V/A/E/R/Y/F, A295S/Y/I/ M/G, G235Y/H and F236I/T/P/M/V/D/S, Y9N, S132K, R145K/P/S/M/Y, L151R/M/A, T178M, "/" means "or "; or the transaminase
- amino acid sequence of the aminotransferase mutant has any combination of mutation sites in the foregoing table.
- a DNA molecule which encodes any of the aforementioned aminotransferase mutants.
- the transaminase mutant encoded by the DNA molecule has good transamination catalytic activity on ketone substrates.
- DNA molecules of the present invention may also exist in the form of "expression cassettes".
- "Expression cassette” refers to a linear or circular nucleic acid molecule, covering DNA and RNA sequences that can direct the expression of a specific nucleotide sequence in an appropriate host cell. Generally speaking, it includes a promoter operatively linked to the target nucleotide, which is optionally operatively linked to a termination signal and/or other regulatory elements.
- the expression cassette may also include sequences required for proper translation of the nucleotide sequence.
- the coding region usually encodes the target protein, but also encodes the target functional RNA in the sense or antisense direction, such as antisense RNA or untranslated RNA.
- the expression cassette containing the target polynucleotide sequence may be chimeric, meaning that at least one of its components is heterologous to at least one of the other components.
- the expression cassette may also be naturally occurring, but obtained by efficient recombination for heterologous expression.
- a recombinant plasmid which contains any of the aforementioned DNA molecules.
- the DNA molecule in the recombinant plasmid is placed in an appropriate position of the recombinant plasmid, so that the DNA molecule can replicate, transcribe or express correctly and smoothly.
- plasmid used in the present invention includes any plasmid, cosmid, bacteriophage or Agrobacterium binary nucleic acid molecule in double-stranded or single-stranded linear or circular form, preferably a recombinant expression plasmid, which may be a prokaryotic expression plasmid or It can be a eukaryotic expression plasmid, but preferably a prokaryotic expression plasmid.
- the recombinant plasmid is selected from pET-22a(+), pET-22b(+), pET-3a(+), pET-3d(+) ), pET-11a(+), pET-12a(+), pET-14b(+), pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+) , PET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a (+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+), pET -38b(+), pET-39b(+), pET-40b(+),
- a host cell in a fourth exemplary embodiment of the present application, contains any of the aforementioned recombinant plasmids.
- Suitable host cells for the present invention include but are not limited to prokaryotic cells, yeast or eukaryotic cells.
- the prokaryotic cells are eubacteria, such as gram-negative bacteria or gram-positive bacteria. More preferably, the prokaryotic cells are E. coli BL21 cells or E. coli DH5 ⁇ competent cells.
- a method for producing chiral amines comprising: using any of the above-mentioned aminotransferase mutants to catalyze ketone compounds Carry out transamination reaction to generate chiral amine compounds
- R 1 and R 2 each independently represent an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group
- R 1 and R 2 can be singly or combined with each other to form a substituted or unsubstituted ring.
- R 1 and R 2 are an optionally substituted or unsubstituted alkyl group having 1 to 20 carbon atoms, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted group.
- R 1 and R 2 are an optionally substituted or unsubstituted alkyl group having 1-10 carbon atoms, an optionally substituted or unsubstituted aralkyl group, or an optional A substituted or unsubstituted aryl group; preferably, substitution refers to a halogen atom, a nitrogen atom, a sulfur atom, a hydroxyl group, a nitro group, a cyano group, a methoxy group, an ethoxy group, a carboxyl group, a carboxymethyl group, a carboxyethyl group Or methylenedioxy substitution.
- the ketone compound is Or it is a substituted or unsubstituted acetophenone compound at any position on the benzene ring, catalyzed by aminotransferase mutants
- Carry out transamination reaction to produce product R represents that any position on the benzene ring is substituted by halogen, nitro, methyl or methoxy; preferably, the halogen is a fluorine atom; more preferably, the fluorine atom is substituted at the meta position of the benzene ring; preferably, the nitro group is substituted on the benzene ring Ortho substitution
- the acetophenone compound is The transaminase mutant of the present application has the highest catalytic activity for the above two acetophenone compounds.
- the mother parent is the mutant SEQ ID NO:1; the conversion rate of the enzyme to the substrate is represented by activity,-represents the initial activity of the mother parent, the multiple of activity increase is represented by +, and + represents an increase of 1-5 Times, ++ means an increase of 5-10 times.
- the above-mentioned mother parent is the mutant SEQ ID NO:1; the level of enzyme conversion rate to the substrate is represented by activity,-represents the initial activity of the mother parent, the multiple of activity increase is represented by +, and + represents an increase of 1-5 times, + + Means an increase of 5-10 times.
- the iterative saturated mutation evolution method can be used to superimpose the mutation sites with increased activity to avoid the evolutionary result being limited to the local highest point instead of reaching the global highest point during the evolution process, and to obtain mutants with increased activity.
- the above-mentioned mother parent is the mutant SEQ ID NO:1; the level of enzyme conversion rate to the substrate is represented by activity,-represents the initial activity of the mother parent, the multiple of activity increase is represented by +, and + represents an increase of 1-5 times, + + Means an increase of 5-10 times, +++ means an increase of 10-20 times, and ++++ means an increase of 20-30 times.
- the above-mentioned mother parent is the mutant SEQ ID NO:1; the level of enzyme conversion rate to the substrate is represented by activity,-represents the initial activity of the mother parent, the multiple of activity increase is represented by +, and + represents an increase of 1-5 times, + + Means an increase of 5-10 times, +++ means an increase of 10-20 times, ++++ means an increase of 20-30 times, +++++ means an increase of 30-50 times, ++++++ means an increase of greater than 50 times.
- the above-mentioned mother parent is the mutant SEQ ID NO:1; the level of enzyme conversion rate to the substrate is represented by activity,-represents the initial activity of the mother parent, the multiple of activity increase is represented by +, and + represents an increase of 1-5 times, + + Means an increase of 5-10 times, +++ means an increase of 10-20 times, ++++ means an increase of 20-30 times, +++++ means an increase of 30-50 times, ++++++ means an increase of greater than 50 times.
- this application uses a method of combining rational design and random mutation to rationally modify the protein of the existing reductase mutant, and The obtained mutants were screened for catalytic activity and stereoselectivity using the substrate ketones of the present application, and finally a mutant strain with high selectivity and high activity was obtained, and the strains containing these mutants were applied to the ketones described in the present application.
- the production efficiency of the corresponding chiral alcohol compound can be improved.
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Abstract
Provided in the present invention are a transaminase mutant and use thereof. Compared with an amino acid sequence shown in SEQ ID NO: 1, an amino acid sequence of the transaminase mutant comprises at least one of the following mutation sites: L166, K149, K146, A168, H73, F133, H82, E24, V194, T294, A295, G235 and F236. The mutant of the present invention has an improved catalytic activity for a transammonization reaction of ketone substrates, and is suitable for industrial production of chiral amines.
Description
本发明涉及酶生产领域，具体而言，涉及一种转氨酶突变体及其应用。The invention relates to the field of enzyme production, in particular to a transaminase mutant and its application.
手性胺是许多重要生物活性分子的结构单元，是合成很多手性药物的关键中间体。有许多重要的神经类药物、心血管药物、抗高血压药物、抗感染药物等都是以手性胺作为中间体来进行合成的。手性胺的制备主要通过化学还原的方法实现，反应中存在反应条件苛刻、使用有毒的过渡态金属催化剂、产品立体选择性低等缺点。当前，在制药产业中利用生物催化法制备手性化合物应用广泛，具有广阔前景。Chiral amines are the structural units of many important biologically active molecules and are key intermediates for the synthesis of many chiral drugs. Many important neurological drugs, cardiovascular drugs, antihypertensive drugs, anti-infective drugs, etc. are synthesized using chiral amines as intermediates. The preparation of chiral amines is mainly achieved through chemical reduction methods. There are disadvantages such as harsh reaction conditions, the use of toxic transition metal catalysts, and low product stereoselectivity. Currently, the use of biocatalysis to prepare chiral compounds in the pharmaceutical industry is widely used and has broad prospects.
ω-转氨酶能够催化酮类底物，通过立体选择性地转氨基作用，高效制备手性胺。因其具有高选择性、高转化率及温和的反应条件等优点，受到研究人员的广泛关注。但在工业化应用时，大多数的野生转氨酶存在催化效率低、立体选择性差、稳定性弱等缺点，使得真正能够被应用的转氨酶并不多。Omega-transaminase can catalyze ketone substrates and efficiently prepare chiral amines through stereoselective transamination. Because of its high selectivity, high conversion rate and mild reaction conditions, it has received extensive attention from researchers. However, in industrial applications, most of the wild transaminases have shortcomings such as low catalytic efficiency, poor stereoselectivity, and weak stability, so that there are not many transaminase that can really be used.
申请公开号为CN107828751A的发明专利申请公开了一种来源于Actinobacteria sp.的ω-转氨酶突变体L107I，该突变体可以高选择性的催化苯乙酮类化合物得到手性氨产品，反应如下：The invention patent application with application publication number CN107828751A discloses an ω-transaminase mutant L107I derived from Actinobacteria sp., which can highly selectively catalyze acetophenone compounds to obtain chiral ammonia products. The reaction is as follows:
但是其初始活性小于10％，活性较低，反应时添加的酶量较多，后处理困难。However, the initial activity is less than 10%, the activity is low, the amount of enzyme added during the reaction is large, and the post-processing is difficult.
发明内容Summary of the invention
本发明的主要目的在于提供一种转氨酶突变体及其应用，以解决现有的转氨酶不适合工业化生产的问题。The main purpose of the present invention is to provide a transaminase mutant and its application to solve the problem that the existing transaminase is not suitable for industrial production.
为了实现上述目的，根据本发明的一个方面，提供了一种转氨酶突变体，与SEQ ID NO:1所示的氨基酸序列相比，该转氨酶突变体的氨基酸序列包括如下至少一个突变位点：L166、K149、K146、A168、H73、F133、H82、E24、V194、T294、A295、G235及F236。In order to achieve the above objective, according to one aspect of the present invention, a transaminase mutant is provided. Compared with the amino acid sequence shown in SEQ ID NO:1, the amino acid sequence of the transaminase mutant includes at least one mutation site as follows: L166 , K149, K146, A168, H73, F133, H82, E24, V194, T294, A295, G235 and F236.
进一步地，转氨酶突变体的氨基酸序列包括如下至少一个突变位点：L166I/V、K149Q/V/C/I/W/M/Y/H/L/F/R、K146R/M/A/P、A168M/V/I/S/P/F、H73T/N/C/Q/R/W/M/K/S、F133S/Q/M/R/A/D、H82S\Q\E\T\Y、E24V/S/A/F、V194I/S/A/H/N、T294Q/W/V/A/E/R/Y/F、A295S/Y/I/M/G、G235Y/H及F236I/T/P/M/V/D/S，Y9N、S132K、R145K/P/S/M/Y、L151R/M/A、T178M,“/”表示“或”；或者转氨酶突变体包括上述突变位点，且与具有突变位点的氨基酸序列有80％以上，优选90％以上，更优选95％以上同一性的氨基酸序列。Furthermore, the amino acid sequence of the aminotransferase mutant includes at least one mutation site as follows: L166I/V, K149Q/V/C/I/W/M/Y/H/L/F/R, K146R/M/A/P , A168M/V/I/S/P/F, H73T/N/C/Q/R/W/M/K/S, F133S/Q/M/R/A/D, H82S\Q\E\T \Y, E24V/S/A/F, V194I/S/A/H/N, T294Q/W/V/A/E/R/Y/F, A295S/Y/I/M/G, G235Y/H And F236I/T/P/M/V/D/S, Y9N, S132K, R145K/P/S/M/Y, L151R/M/A, T178M, "/" means "or"; or aminotransferase mutants include The above-mentioned mutation site is an amino acid sequence that is 80% or more, preferably 90% or more, and more preferably 95% or more identical to the amino acid sequence having the mutation site.
进一步地，转氨酶突变体的氨基酸序列具有如下表中的任一组合的突变位点：Furthermore, the amino acid sequence of the aminotransferase mutant has any combination of mutation sites in the following table:
为了实现上述目的，根据本发明的第二个方面，提供了一种DNA分子，该DNA分子编码上述任一种转氨酶突变体。In order to achieve the above-mentioned object, according to the second aspect of the present invention, a DNA molecule is provided, which encodes any of the above-mentioned aminotransferase mutants.
为了实现上述目的，根据本发明的第三个方面，提供了一种重组质粒，重组质粒连接有上述任一种DNA分子。In order to achieve the above objective, according to the third aspect of the present invention, a recombinant plasmid is provided, which is connected with any of the above-mentioned DNA molecules.
为了实现上述目的，根据本发明的第四个方面，提供了一种宿主细胞，宿主细胞含有上述任一种重组质粒。In order to achieve the above objective, according to the fourth aspect of the present invention, a host cell is provided, which contains any of the above recombinant plasmids.
进一步地，宿主细胞包括原核细胞或真核细胞；优选原核细胞为大肠杆菌。Further, the host cell includes a prokaryotic cell or a eukaryotic cell; preferably, the prokaryotic cell is Escherichia coli.
为了实现上述目的，根据本发明的第五个方面，提供了一种生产手性胺的方法，方法包括：采用上述任一种转氨酶突变体催化酮类化合物
进行转氨反应，生成产物
其中，R
1和R
2分别独立的表示任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基，R
1和R
2可单独或两者互相结合形成取代或未被取代的环。
In order to achieve the above objective, according to the fifth aspect of the present invention, a method for producing chiral amines is provided. The method includes: using any of the above-mentioned aminotransferase mutants to catalyze ketone compounds Carry out transamination reaction to produce product Wherein, R 1 and R 2 each independently represent an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group, R 1 and R 2 can be singly or combined with each other to form a substituted or unsubstituted ring.
进一步地，R
1和R
2为碳原子数为1-20的任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基；优选的，R
1和R
2为碳原子数1-10的任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基；优选的，取代是指被卤素原子、氮原子、硫原子、羟基、硝基、氰基、甲氧基、乙氧基、羧基、羧甲基、羧乙基或亚甲二氧基取代。
Further, R 1 and R 2 are an optionally substituted or unsubstituted alkyl group having 1 to 20 carbon atoms, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group. Group; preferably, R 1 and R 2 are an optionally substituted or unsubstituted alkyl group having 1-10 carbon atoms, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted Aryl; preferably, substitution refers to a halogen atom, a nitrogen atom, a sulfur atom, a hydroxyl group, a nitro group, a cyano group, a methoxy group, an ethoxy group, a carboxyl group, a carboxymethyl group, a carboxyethyl group or a methylenedioxy group replace.
进一步地，酮类化合物为
或者酮类化合物为苯环上任意位置被取代或未被取代的苯乙酮类化合物，转氨酶突变体催化
进行转氨反应，生成产物
R表示苯环上任意位置被卤素、硝基、甲基或甲氧基取代；优选的，卤素为氟原子；更优选氟原子在苯环的间位取代；优选的，硝基在苯环的邻位取代；优选的，苯乙酮类化合物为
Further, the ketone compound is Or the ketone compound is an acetophenone compound substituted or unsubstituted at any position on the benzene ring, catalyzed by the aminotransferase mutant Carry out transamination reaction to produce product R represents that any position on the benzene ring is substituted by halogen, nitro, methyl or methoxy; preferably, the halogen is a fluorine atom; more preferably, the fluorine atom is substituted at the meta position of the benzene ring; preferably, the nitro group is substituted on the benzene ring Ortho substitution; preferably, acetophenone compounds are
应用本发明的技术方案，本发明的突变体，对酮类底物进行转氨反应的催化活性得到提高，适用于手性胺的工业化生产。By applying the technical scheme of the present invention, the mutant of the present invention has improved catalytic activity for transamination of ketone substrates, and is suitable for the industrial production of chiral amines.
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。It should be noted that the embodiments in the application and the features in the embodiments can be combined with each other if there is no conflict. The present invention will be described in detail below in conjunction with embodiments.
“天然存在的”或“野生型”与“突变体”相对，指在自然界中发现的形式。例如，天然存在的或野生型的多肽或多核苷酸序列是存在于生物体的序列，它可以从自然界中的来源中分离，并且没有被人工所特意地修饰或改变。"Naturally occurring" or "wild type" is the opposite of "mutant" and refers to the form found in nature. For example, a naturally-occurring or wild-type polypeptide or polynucleotide sequence is a sequence that exists in an organism, it can be isolated from a source in nature, and has not been intentionally modified or changed by man.
本申请中，涉及例如，细胞、核酸或多肽“重组”时，是指已经以自然界中未存在的方式进行修饰的，或与自然界中存在的形式相同，但是由合成材料和/或通过使用重组技术的处理制备或衍生而得到，或对应于天然或固有形式的细胞、核酸或多肽。其中，非限制性的实例包括在细胞中表达固有(非重组)形式之外的基因或以不同水平表达固有基因的重组细胞。In this application, when referring to, for example, the "recombination" of cells, nucleic acids, or polypeptides, it means that they have been modified in a way that does not exist in nature, or are in the same form as they exist in nature, but are made of synthetic materials and/or through the use of recombinant materials. It is prepared or derived by technological processing, or corresponds to a cell, nucleic acid or polypeptide in a natural or inherent form. Among them, non-limiting examples include recombinant cells expressing genes other than the inherent (non-recombinant) form in cells or expressing inherent genes at different levels.
“序列同一性的百分比”是指多核苷酸序列或氨基酸序列之间的对比，并且通过跨比较窗比较两条最佳比对的序列来确定，其中，多核苷酸序列或氨基酸序列在比较窗中的部分与参考序列相比可以包括添加或缺失(即，空位)，以用于两个序列的最佳比对。百分比可以如 下计算：通过确定两个序列中出现相同的核酸碱基或氨基酸残基的位置的数目，以产生匹配位置的数目，将匹配位置的数目除以比较窗中位置的总数目，并将结果乘以100以得到序列同一性的百分比。可选地，百分比可以如下计算：通过确定两个序列中出现相同的核酸碱基或氨基酸残基或者核酸碱基或氨基酸残基与空位对齐的位置的数目，以产生匹配位置的数目，将匹配位置的数目除以比较窗中位置的总数目，并将结果乘以100以得到序列同一性的百分比。其中，“参考序列”指用作序列比较的基础的指定序列。参考序列可以是更大序列的子集，例如，全长基因或多肽序列的区段。"Percentage of sequence identity" refers to the comparison between polynucleotide sequences or amino acid sequences, and is determined by comparing two optimally aligned sequences across a comparison window, where the polynucleotide sequence or amino acid sequence is in the comparison window. Compared with the reference sequence, the part in may include additions or deletions (ie, gaps) for the best alignment of the two sequences. The percentage can be calculated as follows: by determining the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and The result is multiplied by 100 to get the percentage of sequence identity. Alternatively, the percentage can be calculated as follows: by determining the number of positions where the same nucleic acid base or amino acid residue or the nucleic acid base or amino acid residue is aligned with the gap in the two sequences to generate the number of matching positions, the match Divide the number of positions by the total number of positions in the comparison window and multiply the result by 100 to get the percentage of sequence identity. Here, "reference sequence" refers to a designated sequence used as a basis for sequence comparison. The reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence.
定点突变：是指通过聚合酶链式反应(PCR)等方法向目的DNA片段(可以是基因组，也可以是质粒)中引入所需变化(通常是表征有利方向的变化)，包括碱基的添加、删除、点突变等。定点突变能迅速、高效的提高DNA所表达的目的蛋白的性状及表征，是基因研究工作中一种非常有用的手段。Site-directed mutagenesis: refers to the introduction of desired changes (usually to characterize changes in a favorable direction) into the target DNA fragment (either genome or plasmid) by polymerase chain reaction (PCR) and other methods, including the addition of bases , Deletion, point mutation, etc. Site-directed mutagenesis can quickly and efficiently improve the traits and characterization of the target protein expressed by DNA, which is a very useful method in gene research.
酶的定向进化是一种蛋白质的非理性设计，人为创造特殊的进化条件，模拟自然进化机制，在体外改造基因，应用易错PCR、DNA改组(DNA shuffling)等技术，结合高效筛选***获得具有预期特性的新酶。The directed evolution of enzymes is a kind of irrational design of proteins, artificially creating special evolutionary conditions, simulating natural evolutionary mechanisms, modifying genes in vitro, applying error-prone PCR, DNA shuffling and other technologies, combined with efficient screening systems to obtain New enzymes with expected properties.
为提高现有技术中的转氨酶的酶活性，减少酶的使用量，降低后处理的难度，本申请通过酶进化的方法对来源于Actinobacteria sp.的ω-转氨酶突变体L107I(SEQ ID NO:1)进行进化。In order to improve the enzyme activity of the transaminase in the prior art, reduce the amount of enzyme used, and reduce the difficulty of post-processing, this application uses the method of enzyme evolution to analyze the ω-transaminase mutant L107I (SEQ ID NO:1) derived from Actinobacteria sp. ) To evolve.
来源于Actinobacteria sp.的ω-转氨酶突变体L107I(SEQ ID NO:1)的序列如下：The sequence of the ω-transaminase mutant L107I (SEQ ID NO:1) derived from Actinobacteria sp. is as follows:
以来源于Actinobacteria sp.的ω-转氨酶突变体L107I为模板，设计了17对饱和突变引物(F133、L166、A295、A168、V80、T293、W167、G235、W203、F236、T294、K149、H73、Y78、K146、V194、R145)，利用饱和突变手段，以pET22b(+)为表达载体，获得突变质粒。Using the ω-transaminase mutant L107I from Actinobacteria sp. as a template, 17 pairs of saturated mutation primers (F133, L166, A295, A168, V80, T293, W167, G235, W203, F236, T294, K149, H73, Y78, K146, V194, R145), using saturation mutagenesis means, using pET22b(+) as the expression vector to obtain mutant plasmids.
饱和突变是通过对目的蛋白的编码基因进行改造，短时间内获取靶位点氨基酸分别被其它19种氨基酸替代的突变体的一种方法。此方法不仅是蛋白质定向改造的强有力工具，而且是蛋白质结构－功能关系研究的重要手段。饱和突变往往能获得比单点突变更为理想的进化体。而对于定点突变方法不能解决的这些问题，恰恰是饱和突变方法所擅长的独特之处。Saturation mutation is a method to obtain mutants in which the target amino acids are replaced by 19 other amino acids in a short time by modifying the coding gene of the target protein. This method is not only a powerful tool for directed modification of proteins, but also an important method for the study of protein structure-function relationships. Saturation mutations can often obtain more ideal evolutionary bodies than single point mutations. For these problems that the site-directed mutation method cannot solve, it is precisely the unique point that the saturation mutation method is good at.
在饱和突变获得活性提高的突变体基础上，可对有益的氨基酸位点进行组合，以获得性状更优的突变体。组合突变中双点突变的构建方法和单点突变的构建方法一样，采用全质粒PCR法构建。同时突变2个及以上位点的多点突变通过采用重叠延伸PCR扩增进行，获得含 多点突变的突变基因，两端经限制性内切酶酶切后，连接到表达载体上，转化至大肠杆菌细胞内，涂布于含有100μg/mL氨苄青霉素的LB培养皿中，37℃培养过夜，获得组合突变体，测序鉴定。On the basis of mutants with increased activity obtained by saturation mutations, beneficial amino acid positions can be combined to obtain mutants with better traits. The method for constructing double-point mutations in combinatorial mutations is the same as that for single-point mutations, using the whole plasmid PCR method. Simultaneously mutate two or more sites of multiple-point mutations by using overlap extension PCR amplification to obtain a mutated gene containing multiple-point mutations. After the two ends are digested with restriction enzymes, they are ligated to the expression vector and transformed into The Escherichia coli cells were spread on an LB culture dish containing 100 μg/mL ampicillin and cultured overnight at 37° C. to obtain combined mutants, which were identified by sequencing.
经饱和突变和组合突变获得的突变质粒转化至大肠杆菌细胞内，在大肠杆菌中表达。然后通过超声破碎细胞的方法获得粗酶液。转氨酶诱导表达的最佳条件：25℃，0.2mM IPTG过夜诱导。The mutant plasmid obtained by saturation mutation and combined mutation is transformed into E. coli cells and expressed in E. coli. Then, the crude enzyme solution is obtained by sonicating the cells. The best conditions for inducing expression of transaminase: 25℃, 0.2mM IPTG overnight induction.
获得活性大幅提高的转氨酶突变体后，使用易错PCR的方法对其进行随机突变，构建高质量的突变体库，开发合适的高通量筛选方法，对文库进行筛选，获得活性进一步提高的突变体。After obtaining transaminase mutants with greatly increased activity, they are randomly mutated using error-prone PCR methods to construct a high-quality mutant library, develop appropriate high-throughput screening methods, screen the library, and obtain mutations with further increased activity body.
易错PCR：意为易错条件下的PCR,即容易使复制出的DNA序列出现错误的PCR技术，又称错配PCR或倾向错误PCR。具体是指通过利用低保真度TaqDNA聚合酶和改变PCR反应条件，降低DNA复制的保真度，在新DNA链合成过程中增加碱基错配，从而使扩增产物出现较多点突变的一种体外诱导DNA序列变异的方法。Error-prone PCR: means PCR under error-prone conditions, that is, a PCR technique that easily causes errors in the copied DNA sequence, also known as mismatch PCR or error-prone PCR. Specifically, it refers to the use of low-fidelity TaqDNA polymerase and changing PCR reaction conditions to reduce the fidelity of DNA replication, increase base mismatches during the synthesis of new DNA strands, and cause more point mutations in the amplified products. A method of inducing DNA sequence variation in vitro.
易错PCR是目前最简单、有效的基因体外随机诱变技术，其原理是：碱基的异构为错配提供了可能，组成DNA的4种碱基都有互变异构体存在，其中鸟嘌呤(G)、胞嘧啶(C)和胸腺嘧啶(T)3种含氧碱基有酮式和烯醇式两种互变异构体。腺嘌呤(A)和胸腺嘧啶两种含氮碱基，有胺式、亚胺式两种互变异构体。G、C和T主要以酮式结构存在，烯醇式结构的比率极低，A和T两种含氮碱基上的氮原子主要以氨基(NH2)状态存在，以亚胺基(NH)状态存在的比率极低。不同同分异构体之间氢原子位置的不同及同一位置电子云偏离方向的不同，可使得碱基的配对形式发生改变，这样在复制后的子链上就可能出现错配。例如当胸腺嘧啶以酮式结构存在时，与腺嘌呤配对，而以烯醇式结构存在时，与鸟嘌呤配对，这样就出现了A能配上C，T能配上G的不稳定碱基对，从而造成错配。Error-prone PCR is currently the simplest and most effective in vitro random mutagenesis technique for genes. Its principle is: the isomerization of bases provides the possibility of mismatches. The four bases that make up DNA all have tautomers. The three oxygen-containing bases of guanine (G), cytosine (C) and thymine (T) have two tautomers: keto and enol. Adenine (A) and thymine are two nitrogenous bases, and there are amine and imine tautomers. G, C and T mainly exist as ketone structure, and the ratio of enol structure is extremely low. The nitrogen atoms on the two nitrogen-containing bases of A and T mainly exist in the state of amino (NH2), and in the state of imino (NH). The rate of state existence is extremely low. The difference in the position of the hydrogen atom between different isomers and the deviation of the electron cloud at the same position can change the pairing form of the bases, so that mismatches may appear on the copied daughter strands. For example, when thymine exists in a ketone structure, it pairs with adenine, and when it exists in an enol structure, it pairs with guanine. In this way, there are unstable bases where A can be matched with C and T can be matched with G. Yes, resulting in mismatches.
在已知的几种耐热DNA聚合酶中，TaqDNA聚合酶的错配率最高。Taq DNA聚合酶是发现的耐热DNA聚合酶中活性最高的一种，具有5'-3'外切酶活性，不具3'-5'外切酶活性，因此在合成中对某些单核苷酸错配没有校正功能，所以比有3'-5'校对活性的DNA聚合酶发生错配的概率较高。DNA聚合酶的保真性可以通过多种方法来降低，包括使用4种浓度不同dNTP、添加Mn
2+、提高Mg
2+浓度等。几种诱变方法导致扩增DNA链碱基变异的机理各不相同。MnC1
2是DNA聚合酶的诱变因子，加入Mn
2+可以降低聚合酶对模板的特异性，提高错配率；4种dNTPs浓度的不平衡可以提高碱基错误掺入的概率，实现错配；Mg
2+具有激活Taq酶的作用，增加Mg
2+浓度，使之超过正常用量，能稳定非互补的碱基对；提高Taq DNA聚合酶用量、增加每个循环延伸时间，可以增加错配终端延伸的概率；降低起始模板浓度，会使后面PCR循环的变异模板比例增加。
Among the known heat-resistant DNA polymerases, TaqDNA polymerase has the highest mismatch rate. Taq DNA polymerase is the most active one among the heat-resistant DNA polymerases found. It has 5'-3' exonuclease activity, but not 3'-5' exonuclease activity. Therefore, it has a strong effect on certain mononuclear DNA polymerases during synthesis. There is no correction function for nucleotide mismatches, so the probability of mismatches is higher than that of DNA polymerases with 3'-5' proofreading activity. The fidelity of DNA polymerase can be reduced by a variety of methods, including the use of 4 different concentrations of dNTP, the addition of Mn 2+ , and the increase of Mg 2+ concentration. Several mutagenesis methods lead to different mechanisms of base variation in amplified DNA chains. MnC1 2 is a mutagenic factor of DNA polymerase. Adding Mn 2+ can reduce the specificity of the polymerase to the template and increase the mismatch rate; the unbalanced concentration of 4 dNTPs can increase the probability of base misincorporation and achieve mismatches. ; Mg 2+ has the effect of activating Taq enzyme, increasing the concentration of Mg 2+ to exceed the normal dosage, which can stabilize non-complementary base pairs; increasing the dosage of Taq DNA polymerase and increasing the elongation time of each cycle can increase mismatches Probability of terminal extension; reducing the initial template concentration will increase the proportion of variant templates in subsequent PCR cycles.
因此，在上述研究结果的基础上，申请人提出了本申请的技术方案。在本申请一种典型的实施方式中，提供了一种转氨酶突变体，与SEQ ID NO:1所示的氨基酸序列相比，该转氨 酶突变体的氨基酸序列包括如下至少一种突变位点：L166、K149、K146、A168、H73、F133、H82、E24、V194、T294、A295、G235及F236。本发明的突变体，对酮类底物进行转氨反应的催化活性得到提高，适用于手性胺的工业化生产。Therefore, on the basis of the above-mentioned research results, the applicant proposed the technical solution of this application. In a typical embodiment of the present application, a transaminase mutant is provided. Compared with the amino acid sequence shown in SEQ ID NO:1, the amino acid sequence of the transaminase mutant includes at least one mutation site as follows: L166 , K149, K146, A168, H73, F133, H82, E24, V194, T294, A295, G235 and F236. The mutant of the present invention has improved catalytic activity for transamination of ketone substrates, and is suitable for the industrial production of chiral amines.
在一种优选的实施例中，转氨酶突变体的氨基酸序列还包括如下突变位点之一：L166I/V、K149Q/V/C/I/W/M/Y/H/L/F/R、K146R/M/A/P、A168M/V/I/S/P/F、H73T/N/C/Q/R/W/M/K/S、F133S/Q/M/R/A/D、H82S\Q\E\T\Y、E24V/S/A/F、V194I/S/A/H/N、T294Q/W/V/A/E/R/Y/F、A295S/Y/I/M/G、G235Y/H及F236I/T/P/M/V/D/S，Y9N、S132K、R145K/P/S/M/Y、L151R/M/A、T178M,“/”表示“或”；或者所述转氨酶突变体含有上述突变位点，且与具有上述突变位点的氨基酸序列有80％以上，优选90％以上，更优选95％以上同一性的氨基酸序列。上述转氨酶突变体可以进一步提高转氨反应的催化活性。In a preferred embodiment, the amino acid sequence of the aminotransferase mutant further includes one of the following mutation sites: L166I/V, K149Q/V/C/I/W/M/Y/H/L/F/R, K146R/M/A/P, A168M/V/I/S/P/F, H73T/N/C/Q/R/W/M/K/S, F133S/Q/M/R/A/D, H82S\Q\E\T\Y, E24V/S/A/F, V194I/S/A/H/N, T294Q/W/V/A/E/R/Y/F, A295S/Y/I/ M/G, G235Y/H and F236I/T/P/M/V/D/S, Y9N, S132K, R145K/P/S/M/Y, L151R/M/A, T178M, "/" means "or "; or the transaminase mutant contains the above-mentioned mutation site, and has an amino acid sequence that is more than 80%, preferably more than 90%, and more preferably more than 95% identical to the amino acid sequence having the above-described mutation site. The above-mentioned transaminase mutant can further improve the catalytic activity of the transaminase reaction.
在一种更优选的实施例中，转氨酶突变体的氨基酸序列具有前述表中任一组合的突变位点。In a more preferred embodiment, the amino acid sequence of the aminotransferase mutant has any combination of mutation sites in the foregoing table.
在本申请第二种典型的实施方式中，提供了一种DNA分子，该DNA分子编码上述任一种转氨酶突变体。该DNA分子编码的上述转氨酶突变体对酮类底物具有很好的转氨基催化活性。In the second exemplary embodiment of the present application, a DNA molecule is provided, which encodes any of the aforementioned aminotransferase mutants. The transaminase mutant encoded by the DNA molecule has good transamination catalytic activity on ketone substrates.
本发明的上述DNA分子还可以以“表达盒”的形式存在。“表达盒”是指线性或环状的核酸分子，涵盖了能够指导特定核苷酸序列在恰当宿主细胞中表达的DNA和RNA序列。一般而言，包括与目标核苷酸有效连接的启动子，其任选的是与终止信号和/或其他调控元件有效连接的。表达盒还可以包括核苷酸序列正确翻译所需的序列。编码区通常编码目标蛋白，但在正义或反义方向也编码目标功能RNA，例如反义RNA或非翻译的RNA。包含目标多核苷酸序列的表达盒可以是嵌合的，意指至少一个其组分与其至少一个其他组分是异源的。表达盒还可以是天然存在的，但以用于异源表达的有效重组形成获得的。The above-mentioned DNA molecules of the present invention may also exist in the form of "expression cassettes". "Expression cassette" refers to a linear or circular nucleic acid molecule, covering DNA and RNA sequences that can direct the expression of a specific nucleotide sequence in an appropriate host cell. Generally speaking, it includes a promoter operatively linked to the target nucleotide, which is optionally operatively linked to a termination signal and/or other regulatory elements. The expression cassette may also include sequences required for proper translation of the nucleotide sequence. The coding region usually encodes the target protein, but also encodes the target functional RNA in the sense or antisense direction, such as antisense RNA or untranslated RNA. The expression cassette containing the target polynucleotide sequence may be chimeric, meaning that at least one of its components is heterologous to at least one of the other components. The expression cassette may also be naturally occurring, but obtained by efficient recombination for heterologous expression.
在本申请第三种典型的实施方式中，提供一种重组质粒，该重组质粒含有上述任一种DNA分子。上述重组质粒中的DNA分子置于重组质粒的适当位置，使得上述DNA分子能够正确地、顺利地复制、转录或表达。In a third exemplary embodiment of the present application, a recombinant plasmid is provided, which contains any of the aforementioned DNA molecules. The DNA molecule in the recombinant plasmid is placed in an appropriate position of the recombinant plasmid, so that the DNA molecule can replicate, transcribe or express correctly and smoothly.
虽然本发明在限定上述DNA分子时所用限定语为“含有”，但其并不意味着可以在DNA序列的两端任意加入与其功能不相关的其他序列。本领域技术人员知晓，为了满足重组操作的要求，需要在DNA序列的两端添加合适的限制性内切酶的酶切位点，或者额外增加启动密码子、终止密码子等，因此，如果用封闭式的表述来限定将不能真实地覆盖这些情形。Although the qualifier used in the present invention to define the above-mentioned DNA molecule is "containing", it does not mean that other sequences that are not related to its function can be arbitrarily added to both ends of the DNA sequence. Those skilled in the art know that in order to meet the requirements of recombination operations, it is necessary to add appropriate restriction endonuclease cleavage sites at both ends of the DNA sequence, or to add additional start codons, stop codons, etc., therefore, if you use Closed expressions to limit will not truly cover these situations.
本发明中所使用的术语“质粒”包括双链或单链线状或环状形式的任何质粒、粘粒、噬菌体或农杆菌二元核酸分子，优选为重组表达质粒，可以是原核表达质粒也可以是真核表达质粒，但优选原核表达质粒，在某些实施方案中，重组质粒选自pET-22a(+)、pET-22b(+)、pET-3a(+)、pET-3d(+)、pET-11a(+)、pET-12a(+)、pET-14b(+)、pET-15b(+)、pET-16b(+)、pET-17b(+)、pET-19b(+)、pET-20b(+)、pET-21a(+)、pET-23a(+)、pET-23b(+)、pET-24a (+)、pET-25b(+)、pET-26b(+)、pET-27b(+)、pET-28a(+)、pET-29a(+)、pET-30a(+)、pET-31b(+)、pET-32a(+)、pET-35b(+)、pET-38b(+)、pET-39b(+)、pET-40b(+)、pET-41a(+)、pET-41b(+)、pET-42a(+)、pET-43a(+)、pET-43b(+)、pET-44a(+)、pET-49b(+)、pQE2、pQE9、pQE30、pQE31、pQE32、pQE40、pQE70、pQE80、pRSET-A、pRSET-B、pRSET-C、pGEX-5X-1、pGEX-6p-1、pGEX-6p-2、pBV220、pBV221、pBV222、pTrc99A、pTwin1、pEZZ18、pKK232-18、pUC-18或pUC-19。更优选，上述重组质粒是pET-22b(+)。The term "plasmid" used in the present invention includes any plasmid, cosmid, bacteriophage or Agrobacterium binary nucleic acid molecule in double-stranded or single-stranded linear or circular form, preferably a recombinant expression plasmid, which may be a prokaryotic expression plasmid or It can be a eukaryotic expression plasmid, but preferably a prokaryotic expression plasmid. In some embodiments, the recombinant plasmid is selected from pET-22a(+), pET-22b(+), pET-3a(+), pET-3d(+) ), pET-11a(+), pET-12a(+), pET-14b(+), pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+) , PET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a (+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+), pET -38b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42a(+), pET-43a(+), pET- 43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X -1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18 or pUC-19. More preferably, the aforementioned recombinant plasmid is pET-22b(+).
在本申请第四种典型的实施方式中，提供了一种宿主细胞，该宿主细胞含有上述任一种重组质粒。适用于本发明的宿主细胞包括但不仅限于原核细胞、酵母或真核细胞。优选原核细胞为真细菌，例如革兰氏阴性菌或革兰氏阳性菌。更优选原核细胞为大肠杆菌BL21细胞或大肠杆菌DH5α感受态细胞。In a fourth exemplary embodiment of the present application, a host cell is provided, and the host cell contains any of the aforementioned recombinant plasmids. Suitable host cells for the present invention include but are not limited to prokaryotic cells, yeast or eukaryotic cells. Preferably, the prokaryotic cells are eubacteria, such as gram-negative bacteria or gram-positive bacteria. More preferably, the prokaryotic cells are E. coli BL21 cells or E. coli DH5α competent cells.
在本申请第五种典型的实施方式中，提供一种生产手性胺的方法，该方法包括：采用上述任一种转氨酶突变体催化酮类化合物
进行转氨反应，生成手性胺化合物
其中，R
1和R
2分别独立的表示任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基，R
1和R
2可单独或两者互相结合形成取代或未被取代的环。
In a fifth exemplary embodiment of the present application, a method for producing chiral amines is provided, the method comprising: using any of the above-mentioned aminotransferase mutants to catalyze ketone compounds Carry out transamination reaction to generate chiral amine compounds Wherein, R 1 and R 2 each independently represent an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group, R 1 and R 2 can be singly or combined with each other to form a substituted or unsubstituted ring.
在一种优选的实施例中，R
1和R
2为碳原子数为1-20的任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基；优选的，R
1和R
2为碳原子数1-10的任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基；优选的，取代是指被卤素原子、氮原子、硫原子、羟基、硝基、氰基、甲氧基、乙氧基、羧基、羧甲基、羧乙基或亚甲二氧基取代。
In a preferred embodiment, R 1 and R 2 are an optionally substituted or unsubstituted alkyl group having 1 to 20 carbon atoms, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted group. Or an unsubstituted aryl group; preferably, R 1 and R 2 are an optionally substituted or unsubstituted alkyl group having 1-10 carbon atoms, an optionally substituted or unsubstituted aralkyl group, or an optional A substituted or unsubstituted aryl group; preferably, substitution refers to a halogen atom, a nitrogen atom, a sulfur atom, a hydroxyl group, a nitro group, a cyano group, a methoxy group, an ethoxy group, a carboxyl group, a carboxymethyl group, a carboxyethyl group Or methylenedioxy substitution.
在一种更优选的实施例中，酮类化合物为为
或者为苯环上任意位置被取代或未被取代的苯乙酮类化合物，转氨酶突变体催化
进行转氨反应，生 成产物
R表示苯环上任意位置被卤素、硝基、甲基或甲氧基取代；优选的，卤素为氟原子；更优选氟原子在苯环的间位取代；优选的，硝基在苯环的邻位取代；
In a more preferred embodiment, the ketone compound is Or it is a substituted or unsubstituted acetophenone compound at any position on the benzene ring, catalyzed by aminotransferase mutants Carry out transamination reaction to produce product R represents that any position on the benzene ring is substituted by halogen, nitro, methyl or methoxy; preferably, the halogen is a fluorine atom; more preferably, the fluorine atom is substituted at the meta position of the benzene ring; preferably, the nitro group is substituted on the benzene ring Ortho substitution
在一种更优选的实施例中，苯乙酮类化合物为
本申请的转氨酶突变体对上述两种苯乙酮类化合物的催化活性最高。
In a more preferred embodiment, the acetophenone compound is The transaminase mutant of the present application has the highest catalytic activity for the above two acetophenone compounds.
下面将结合具体的实施例来进一步说明本申请的有益效果。The following will further illustrate the beneficial effects of the present application in conjunction with specific embodiments.
下述实施例提到的原料为：The raw materials mentioned in the following examples are:
原料1：Raw material 1:
原料2：Raw material 2:
原料3：Raw material 3:
原料4：Raw material 4:
实施例1Example 1
5mL的反应瓶中分别加入30mg原料1、原料2、原料3和原料4，加入pH 8.5的Tris-Cl(0.1M)，181μL的异丙胺盐酸盐(6M)，0.9mg的PLP，加入转氨酶300mg(具体见表1)，混匀，总体积1500μL，于30℃，200rpm摇床反应16h。反应结束后，向反应体系中加2倍体积的甲醇，混匀，12000rpm，离心3min，取上清，HPLC检测，波长210nm。部分突变体反应特性如下：Add 30mg of raw material 1, raw material 2, raw material 3, and raw material 4 to a 5mL reaction flask, add Tris-Cl (0.1M) with pH 8.5, 181μL of isopropylamine hydrochloride (6M), 0.9mg of PLP, and add transaminase 300 mg (see Table 1 for details), mix well, and the total volume is 1500 μL, and react at 30° C., 200 rpm shaker for 16 hours. After the reaction, add 2 times the volume of methanol to the reaction system, mix well, centrifuge at 12000 rpm for 3 min, take the supernatant, and detect by HPLC with a wavelength of 210 nm. The response characteristics of some mutants are as follows:
表1：Table 1:
上表中，母本为突变体SEQ ID NO:1；酶对底物转化率的高低用活性来表示，-表示母本的初始活性，活性提高的倍数用+表示，+表示提高1-5倍，++表示提高5-10倍。In the above table, the mother parent is the mutant SEQ ID NO:1; the conversion rate of the enzyme to the substrate is represented by activity,-represents the initial activity of the mother parent, the multiple of activity increase is represented by +, and + represents an increase of 1-5 Times, ++ means an increase of 5-10 times.
由表1的结果可以看出，单点突变体的转化效果较母本有所提高，但并未达到最理想的效果。对有益突变位点进行组合，可进一步提高突变体活性。From the results in Table 1, it can be seen that the transformation effect of the single-point mutant is higher than that of the female parent, but it has not reached the optimal effect. Combining beneficial mutation sites can further improve mutant activity.
实施例2：Example 2:
5mL的反应瓶中分别加入30mg原料1、原料2、原料3和原料4，加入pH 8.5的Tris-Cl(0.1M)，181μL的异丙胺盐酸盐(6M)，0.9mg的PLP，加入转氨酶210mg(具体见表2)，混匀，总体积1500μL，于30℃，200rpm摇床反应16h。反应结束后，向反应体系中加2倍体积的甲醇，混匀，12000rpm，离心3min，取上清，HPLC检测，波长210nm。部分突变体反应特性如下：Add 30mg of raw material 1, raw material 2, raw material 3, and raw material 4 to a 5mL reaction flask, add Tris-Cl (0.1M) with pH 8.5, 181μL of isopropylamine hydrochloride (6M), 0.9mg of PLP, and add transaminase 210 mg (see Table 2 for details), mix well, and the total volume is 1500 μL, and react at 30° C. and 200 rpm shaker for 16 hours. After the reaction, add 2 times the volume of methanol to the reaction system, mix well, centrifuge at 12000 rpm for 3 min, take the supernatant, and detect by HPLC with a wavelength of 210 nm. The response characteristics of some mutants are as follows:
表2：Table 2:
上述母本为突变体SEQ ID NO:1；酶对底物转化率的高低用活性来表示，-表示母本的初始活性，活性提高的倍数用+表示，+表示提高1-5倍，++表示提高5-10倍。The above-mentioned mother parent is the mutant SEQ ID NO:1; the level of enzyme conversion rate to the substrate is represented by activity,-represents the initial activity of the mother parent, the multiple of activity increase is represented by +, and + represents an increase of 1-5 times, + + Means an increase of 5-10 times.
由表2结果可以看出，突变体活性进一步提高，但仍未达到最佳的效果。可通过迭代饱和突变的进化方法，叠加活性提高的突变位点，避免进化过程中，进化结果只限于局部最高点而不能到达全局最高点，获得活性提高的突变体。It can be seen from the results in Table 2 that the activity of the mutant is further improved, but the optimal effect is still not achieved. The iterative saturated mutation evolution method can be used to superimpose the mutation sites with increased activity to avoid the evolutionary result being limited to the local highest point instead of reaching the global highest point during the evolution process, and to obtain mutants with increased activity.
实施例3Example 3
5mL的反应瓶中分别加入30mg原料1、原料2、原料3和原料4，加入pH 8.5的Tris-Cl(0.1M)，181μL的异丙胺盐酸盐(6M)，0.9mg的PLP，加入转氨酶60mg(具体见表3)，混匀，总体积900μL，于30℃，200rpm摇床反应16h。反应结束后，向反应体系中加2倍体积的甲醇，混匀，12000rpm，离心3min，取上清，HPLC检测，波长210nm。部分突变体反应特性如下：Add 30mg of raw material 1, raw material 2, raw material 3, and raw material 4 to a 5mL reaction flask, add Tris-Cl (0.1M) with pH 8.5, 181μL of isopropylamine hydrochloride (6M), 0.9mg of PLP, and add transaminase 60 mg (see Table 3 for details), mix well, the total volume is 900 μL, and react at 30° C., 200 rpm shaker for 16 hours. After the reaction is over, add 2 times the volume of methanol to the reaction system, mix well, centrifuge at 12000 rpm for 3 min, take the supernatant, and detect by HPLC with a wavelength of 210 nm. The response characteristics of some mutants are as follows:
表3：table 3:
上述母本为突变体SEQ ID NO:1；酶对底物转化率的高低用活性来表示，-表示母本的初始活性，活性提高的倍数用+表示，+表示提高1-5倍，++表示提高5-10倍，+++表示提高10-20倍，++++表示提高20-30倍。The above-mentioned mother parent is the mutant SEQ ID NO:1; the level of enzyme conversion rate to the substrate is represented by activity,-represents the initial activity of the mother parent, the multiple of activity increase is represented by +, and + represents an increase of 1-5 times, + + Means an increase of 5-10 times, +++ means an increase of 10-20 times, and ++++ means an increase of 20-30 times.
实施例4Example 4
5mL的反应瓶分别中加入30mg原料1、原料2、原料3和原料4，加入pH 8.5的Tris-Cl(0.1M)，181μL的异丙胺盐酸盐(6M)，0.9mg的PLP，加入转氨酶60mg(具体见表4)，混匀，总体积900μL，于30℃，200rpm摇床反应16h。反应结束后，向反应体系中加2倍体积的甲醇，混匀，12000rpm，离心3min，取上清，HPLC检测，波长210nm。部分突变体反应特性如下：Add 30mg of raw material 1, raw material 2, raw material 3, and raw material 4 to a 5 mL reaction flask, add Tris-Cl (0.1M) with pH 8.5, 181 μL of isopropylamine hydrochloride (6M), 0.9 mg of PLP, and add transaminase 60 mg (see Table 4 for details), mix well, the total volume is 900 μL, and react at 30° C., 200 rpm shaker for 16 hours. After the reaction, add 2 times the volume of methanol to the reaction system, mix well, centrifuge at 12000 rpm for 3 min, take the supernatant, and detect by HPLC with a wavelength of 210 nm. The response characteristics of some mutants are as follows:
表4：Table 4:
上述母本为突变体SEQ ID NO:1；酶对底物转化率的高低用活性来表示，-表示母本的初始活性，活性提高的倍数用+表示，+表示提高1-5倍，++表示提高5-10倍，+++表示提高10-20倍，++++表示提高20-30倍，+++++表示提高30-50倍，++++++表示提高大于50倍。The above-mentioned mother parent is the mutant SEQ ID NO:1; the level of enzyme conversion rate to the substrate is represented by activity,-represents the initial activity of the mother parent, the multiple of activity increase is represented by +, and + represents an increase of 1-5 times, + + Means an increase of 5-10 times, +++ means an increase of 10-20 times, ++++ means an increase of 20-30 times, +++++ means an increase of 30-50 times, ++++++ means an increase of greater than 50 times.
由表4结果可以看出，运用迭代饱和突变使得突变体活性大幅提高。下一步可继续通过随机突变(易错PCR)，进一步提高突变体活性，得到可用于工业生产的最终突变体。It can be seen from the results in Table 4 that the use of iterative saturation mutation greatly increases the activity of the mutant. In the next step, random mutation (error-prone PCR) can be continued to further improve the activity of the mutant and obtain the final mutant that can be used in industrial production.
实施例5Example 5
5mL的反应瓶中分别加入30mg原料1、原料2、原料3和原料4，加入pH 8.5的Tris-Cl(0.1M)，181μL的异丙胺盐酸盐(6M)，0.9mg的PLP，加入转氨酶30mg(具体见表5)，混匀，总体积900μL，于30℃，200rpm摇床反应16h。反应结束后，向反应体系中加2倍体积的甲醇，混匀，12000rpm，离心3min，取上清，HPLC检测，波长210nm。部分突变体反应特性如下：Add 30mg of raw material 1, raw material 2, raw material 3, and raw material 4 to a 5mL reaction flask, add Tris-Cl (0.1M) with pH 8.5, 181μL of isopropylamine hydrochloride (6M), 0.9mg of PLP, and add transaminase 30 mg (see Table 5 for details), mix well, the total volume is 900 μL, and react at 30° C. and 200 rpm shaker for 16 hours. After the reaction, add 2 times the volume of methanol to the reaction system, mix well, centrifuge at 12000 rpm for 3 min, take the supernatant, and detect by HPLC with a wavelength of 210 nm. The response characteristics of some mutants are as follows:
表5：table 5:
上述母本为突变体SEQ ID NO:1；酶对底物转化率的高低用活性来表示，-表示母本的初始活性，活性提高的倍数用+表示，+表示提高1-5倍，++表示提高5-10倍，+++表示提高10-20倍，++++表示提高20-30倍，+++++表示提高30-50倍，++++++表示提高大于50倍。The above-mentioned mother parent is the mutant SEQ ID NO:1; the level of enzyme conversion rate to the substrate is represented by activity,-represents the initial activity of the mother parent, the multiple of activity increase is represented by +, and + represents an increase of 1-5 times, + + Means an increase of 5-10 times, +++ means an increase of 10-20 times, ++++ means an increase of 20-30 times, +++++ means an increase of 30-50 times, ++++++ means an increase of greater than 50 times.
实施例6Example 6
5mL的反应瓶中分别加入30mg原料1、原料2、原料3和原料4，加入pH 8.5的Tris-Cl(0.1M)，181μL的异丙胺盐酸盐(6M)，0.9mg的PLP，加入转氨酶30mg(具体见表6)，混匀，总体积900μL，于30℃，200rpm摇床反应16h。反应结束后，向反应体系中加入2mL乙酸乙酯，12000rpm，离心3min，进行萃取，取上清，HPLC检测ee值。突变体反应特性如下：Add 30mg of raw material 1, raw material 2, raw material 3, and raw material 4 to a 5mL reaction flask, add Tris-Cl (0.1M) with pH 8.5, 181μL of isopropylamine hydrochloride (6M), 0.9mg of PLP, and add transaminase 30 mg (see Table 6 for details), mix well, the total volume is 900 μL, and react at 30° C. and 200 rpm shaker for 16 hours. After the reaction, 2 mL of ethyl acetate was added to the reaction system, centrifuged at 12000 rpm for 3 min, and extracted, the supernatant was taken, and the ee value was detected by HPLC. The response characteristics of the mutant are as follows:
表6：Table 6:
实施例7Example 7
2L的反应瓶中分别加入20g原料1和原料2，加入pH 8.5的Tris-Cl(0.1M)，120mL的异丙胺盐酸盐(6M)，0.6g的PLP，加入转氨酶20g(具体见表6)，混匀，总体积600mL，于30℃，200rpm摇床反应16h。反应结束后，向反应体系中加入600mL乙酸乙酯，12000rpm，离心3min，进行萃取。样品经过后处理，HPLC检测，测定收率、纯度及ee值。Add 20g of raw material 1 and raw material 2 to a 2L reaction flask, add Tris-Cl (0.1M) of pH 8.5, 120mL of isopropylamine hydrochloride (6M), 0.6g of PLP, and add 20g of transaminase (see Table 6 for details) ), mix well, with a total volume of 600 mL, and react at 30°C with a shaker at 200 rpm for 16 hours. After the completion of the reaction, 600 mL of ethyl acetate was added to the reaction system at 12000 rpm and centrifuged for 3 min for extraction. The sample is post-processed and tested by HPLC to determine the yield, purity and ee value.
表7：Table 7:
从以上的描述中，可以看出，本发明上述的实施例实现了如下技术效果：本申请通过理性设计和随机突变相结合的方法对已有的还原酶突变体的蛋白进行理性改造，并对获得的突变体使用本申请的底物酮进行催化活力和立体选择性筛选，最终获得了高选择性和高活力的突变体菌株，将含有这些突变体的菌株应用于本申请所述的酮类底物的催化还原反应中，能够提高其相应的手性醇类化合物的生成效率。From the above description, it can be seen that the above-mentioned embodiments of the present invention have achieved the following technical effects: this application uses a method of combining rational design and random mutation to rationally modify the protein of the existing reductase mutant, and The obtained mutants were screened for catalytic activity and stereoselectivity using the substrate ketones of the present application, and finally a mutant strain with high selectivity and high activity was obtained, and the strains containing these mutants were applied to the ketones described in the present application. In the catalytic reduction reaction of the substrate, the production efficiency of the corresponding chiral alcohol compound can be improved.
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above are only preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
- 一种转氨酶突变体，其特征在于，所述转氨酶突变体的氨基酸序列与SEQ ID NO:1所示的氨基酸序列相比，在如下任一个位点发生突变：L166I/V、K149Q/V/C/I/W、K146R/M/A、A168M/V/I、H73T/N/C/Q、F133S/Q/M,“/”表示“或”；或者在如下任一组合位点发生突变：A transaminase mutant, characterized in that the amino acid sequence of the transaminase mutant is compared with the amino acid sequence shown in SEQ ID NO:1 and has a mutation at any of the following positions: L166I/V, K149Q/V/C /I/W, K146R/M/A, A168M/V/I, H73T/N/C/Q, F133S/Q/M, "/" means "or"; or a mutation occurs at any of the following combinations:
- 一种DNA分子，其特征在于，所述DNA分子编码权利要求1所述的转氨酶突变体。A DNA molecule, characterized in that it encodes the aminotransferase mutant of claim 1.
- 一种重组质粒，其特征在于，所述重组质粒连接有权利要求2所述的DNA分子。A recombinant plasmid, characterized in that the DNA molecule according to claim 2 is connected to the recombinant plasmid.
- 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求3所述的重组质粒。A host cell, characterized in that the host cell contains the recombinant plasmid of claim 3.
- 根据权利要求4所述的宿主细胞，其特征在于，所述宿主细胞包括原核细胞或真核细胞；所述原核细胞为大肠杆菌。The host cell of claim 4, wherein the host cell comprises a prokaryotic cell or a eukaryotic cell; the prokaryotic cell is Escherichia coli.
- 一种生产手性胺的方法，其特征在于，所述方法包括：采用权利要求1所述的转氨酶突变体催化酮类化合物进行转氨反应生成产物，A method for producing chiral amines, characterized in that the method comprises: using the aminotransferase mutant of claim 1 to catalyze a ketone compound to undergo a transamination reaction to produce a product,所述酮类化合物为或者为苯乙酮类化合物
其中，R表示苯环上任意位置被卤素或硝基取代。 The ketone compound is
Or acetophenone compoundsWherein, R represents that any position on the benzene ring is substituted by a halogen or a nitro group. - 根据权利要求6所述的方法，其特征在于，The method of claim 6, wherein:所述卤素为氟原子。The halogen is a fluorine atom.
- 根据权利要求6所述的方法，其特征在于，The method of claim 6, wherein:所述苯乙酮类化合物为The acetophenone compound is
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| EP20933800.3A EP4144837A4 (en) | 2020-04-29 | 2020-05-29 | Transaminase mutant and use thereof |
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| CN114181918B (en) * | 2020-12-04 | 2023-09-15 | 浙江科技学院 | Omega-aminotransferase mutant obtained by DNA synthesis shuffling combination mutation and application |
| CN115873910B (en) * | 2023-02-28 | 2023-07-28 | 凯莱英生命科学技术(天津)有限公司 | Application of aminotransferase in catalytic synthesis of lactam compound and synthesis method of lactam compound |
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| CN107805648A (en) * | 2017-10-10 | 2018-03-16 | 凯莱英生命科学技术(天津)有限公司 | The method for preparing the amines with multiple chiral centers |
| CN107828751A (en) | 2017-11-06 | 2018-03-23 | 凯莱英生命科学技术(天津)有限公司 | Transaminase mutant and its application |
| CN108823179A (en) * | 2018-06-30 | 2018-11-16 | 浙江工业大学 | A kind of transaminase from actinomyces, mutant, recombinant bacterium and application |
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| CN109234327A (en) | 2017-07-11 | 2019-01-18 | 上海弈柯莱生物医药科技有限公司 | A kind of application of the transaminase of stereoselectivity in asymmetric syntheses Chiral Amine |
| JP7022819B2 (en) * | 2017-11-06 | 2022-02-18 | アシムケム ライフ サイエンス （ティエンジン） カンパニー リミテッド | Transaminase mutants and their use |
| CN108048419B (en) * | 2017-11-15 | 2020-10-23 | 凯莱英生命科学技术(天津)有限公司 | Transaminase mutants and uses thereof |
| ES2816102T3 (en) * | 2017-11-17 | 2021-03-31 | Enzymicals Ag | Procedure for the preparation of amines from carbonyl compounds by transaminase reaction under salt precipitation |
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| TANG XIAO-LING; ZHANG NAN-NAN; YE GUO-YAN; ZHENG YU-GUO: "Efficient biosynthesis of (R)-3-amino-1-butanol by a novel (R)-selective transaminase fromActinobacteriasp.", JOURNAL OF BIOTECHNOLOGY, ELSEVIER, AMSTERDAM NL, vol. 295, 1 January 1900 (1900-01-01), Amsterdam NL , pages 49 - 54, XP085641894, ISSN: 0168-1656, DOI: 10.1016/j.jbiotec.2019.02.008 * |
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| US20230235300A1 (en) | 2023-07-27 |
| JP7462795B2 (en) | 2024-04-05 |
| JP2023523362A (en) | 2023-06-02 |
| CN111235127A (en) | 2020-06-05 |
| CN111235127B (en) | 2020-08-14 |
| EP4144837A1 (en) | 2023-03-08 |
| KR20220161459A (en) | 2022-12-06 |
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